The ischemia/reperfusion (I/R) injury results in loss of microtubules (MTs) and F-actin in kidney proximal tubule cells. Using a model of mild ischemia (30-minute unilateral clamp of renal vessels) we have shown that those cytoskeletal elements are mostly repolymerized by day 3 after I/R, but polarization of the plasma membrane becomes normal only by day 5-7, which parallels the normalization of kidney functional parameters. The observation of abnormal MT polarity during the late phase of the recovery after I/R, lead us to hypothesize that Microtubule-Organizing Centers (MTOCs) are redistributed at that time because an MTOC component, GCP6, which mediates attachment of MTOCs to intermediate filaments, is phosphorylated in a regulatory cassette located between aa 1395 and 1399. Therefore, I propose the following specific aims to test this hypothesis: 1. Determine the role of GCP6 in the attachment of MTOCs to keratin intermediate filaments under the apical domain of simple epithelial cells. 1.1 Determine the binding domains in GCP6 for keratins 8/19 using deletion mutants. 1.2 Define the binding domains in keratin 8 for GCP6 using the same method. 1.3 Over-express the mutants mentioned above to identify dominant-negative effects on the localization of MTOCs. 2. Characterize the phosphorylation of GCP6 by kinases known to block the attachment of MTOCs to keratins. 2.1 Identify the p34cdc and plkl phosphorylation sites. 2.2 Obtain S->D and S->A mutants of GCP6 in that site and verify changes in keratin 8 binding and response to p34cc/c2 signaling. 2.3 Overexpress those mutants and analyze their localization and ability to organize the normal architecture of MTs. 3. Identify phosphorylation sites of GCP6 modified in vivo after I/R that account detachment of MTOCs during days 3-5 after I/R in proximal tubule cells and determine the consequences of a disarray of MTOCs on epithelial polarity. 3.1 Determine the kinetics of phosphorylation of GCP6-S1396 in post-ischemic kidney using a specific anti-phosphopeptide antibody, localize phospho-81396 GCP6, and determine the effect of specific blockers of GSK3 on MTOC distribution after I/R in vivo. 3.2 Obtain an S->D mutant of the phospho-site studied en 3.1 (possibly the GSK-3 site nearby S1397), and verify lack of binding to keratins and the effect on MTOC localization in vivo. 3.3 Assess depolarization of apical plasma membrane proteins, changes in vectorial membrane delivery and tight junction function after blocking the attachment of MTOCs to apical IPs. The long term goal of the project is to characterize a signaling pathway responsible for the depolarization of MTOCs and plasma membrane proteins in the late stages of the recovery after I/R and to design an intervention to revert it and improve recovery times after I/R. Lay statement: Acute renal failure affects 5% of patients admitted in hospitals and is a leading cause for the need of kidney transplants. In this project we analyze the structure and signaling that organizes the cytoskeleton during the second half of the recovery and ways to possibly shorten the period of kidney failure and to improve its recovery. The concepts can also be applied to the recovery of kidney transplants, that also suffer the consequences of temporal lack of blood supply. [unreadable] [unreadable] [unreadable]